Integrated nucleic acid analysis

ABSTRACT

The present invention relates to an integrated method of nucleic acid analysis, and more particularly to a simplified sample pre-treatment, which renders the method more easily automated, where the sample is provided on or applied onto a solid matrix and the subsequent amplification and detection steps are performed in one single, sealed reaction vial without removing the matrix.

FIELD OF THE INVENTION

The present invention relates to an integrated method of nucleic acidanalysis, and more particularly to a simplified sample pre-treatment,which renders the method more easily automated. The method of thepresent invention provides minimized risks related to contamination dueto minimal handling of the samples.

BACKGROUND OF THE INVENTION

As a result of the rapidly developing genomic testing, whole nucleicacid analysis is an increasing task in many genetic laboratories. Thepolymerase chain reaction (PCR) is a well established method foramplifying nucleic acid sequences, and the method is routinely used innumerous application areas, such as microbiological testing, expressionstudies, determination of genetic variation in population, and genetictesting, forensics and food and environmental testing. Testing ofnucleic acids using PCR generally involves three steps: samplepreparation, amplification and detection. However, the processes forperforming the tests used today are often laborious. The current trendis towards simplified assays allowing automation for nucleic acidanalysis.

The increased number of tests to be run raises a need for cost-effectiveoperations based on integration and automation of the assay procedures,and the automation of the whole DNA analysis is an increasing task inmany genetic laboratories. Although several attempts to performDNA-analysis as high throughput assays has been described, the logisticsof sample handling from sample preparation and pre-treatment to theultimate analysis still requires manual handling and physicaltransportation of the samples. In addition, the need to physicallytransfer amplified DNA samples within the lab poses a seriouscontamination risk.

One type of a simplified assay that reduces sample handling is providedby so-called closed-tube assays. In closed-tube assays, the PCR productis analyzed in the amplification tube by a homogeneous method, such asTaqMan® (U.S. Pat. No. 5,210,015) or by molecular beacons (U.S. Pat. No.5,118,801). These approaches allow integration of amplification anddetection. However, they lack integration of sample preparation withamplification and detection.

Pre-treatment of the sample is required to remove common inhibitors tonucleic acid amplification that may be present in samples frombiological sources. Inhibitors to amplification include, e.g. naturallyoccurring chelating agents, enzymes and/or proteins that can damageeither nucleic acid templates or PCR polymerases used in theamplification reactions. In addition, the common anticoagulants that areused to treat whole blood samples can interfere with nucleic acidamplification reactions.

Numerous technologies have been developed to purify nucleic acids frombiological samples but all available procedures are time-consuming andlabour intensive. There are several automated stations for samplepreparation available on the market, based on silica-chaotrop extraction(U.S. Pat. No. 5,234,809) columns, such as Q IAamp®, or the like, andvarious magnetic bead systems. A different approach is the FTA®Technology (U.S. Pat. No. 5,496,562) that lyses cell membranes as soonas the sample is applied onto a coated filtration matrix allowingimmobilization of nucleic acids onto the matrix. After washing, thenucleic acids can be released in a manner that enables them to beamplified by PCR.

Another approach is represented by the development of different solidmatrices for collecting, transporting, storing and purifying biologicalsamples, such as clinical whole blood, saliva or faecal samples, fornucleic acid analysis. U.S. Pat. No. 5,807,527 describes a solid mediumfor long term storage of blood DNA, which comprises a composition, whichprotects against degradation of DNA, a protein denaturing agent and afree radical trap.

Another example of this approach is the described in U.S. Pat. No.5,939,259, which discloses an absorbent material, which does not bindnucleic acids irreversibly which is impregnated with a chaotropic salt.

In these known technologies, the DNA of stored blood samples isextracted from the medium before performing PCR, or the DNA is used inPCR in situ on the solid medium after extensive purification, asdescribed in U.S. Pat. No. 5,807,527. Extraction or elution of DNA fromthe medium requires a multi-step procedure with special solutions andincubations.

Known technologies using solid media for collecting, storing andpurifying DNA thus involve multi-step procedures, some using severalseparate vials and solutions, for generating a sample useful foramplification and detection. Moreover, certain of these methodsunnecessarily produce waste.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical presentation of the result of the comparative testdescribed in Example 1, analyzing the effect of pre-washing driedsamples on collection paper.

FIG. 2 is a graphical presentation of the result of the test describedin Example 2, where the release of DNA from dried samples, amplificationand detection of amplified DNA is performed in a single reaction vial.

FIG. 3 is a graphical presentation of the result of the test describedin Example 3, where the release of DNA from dried samples, amplificationand detection of amplified DNA is performed in a single reaction vial,and where all necessary reagents were added simultaneously.

FIG. 4 is a graphical presentation of the result of the test describedin Example 4, where a whole blood sample is applied to an absorbentmatrix in the reaction vial, and amplification and detection ofamplified DNA is performed in the same reaction vial.

FIG. 5 is a graphical presentation of the result of the test describedin Example 5, where all necessary reagents were provided in dried formin the reaction vial.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a method for analyzing a target nucleicacid in a biological sample, comprising the following steps of i)providing an absorbent matrix in a reaction vial; ii) providing abiological sample containing said target nucleic acids on or applying itonto said absorbent matrix; iii) providing in said reaction vial areagent mixture for amplifying said nucleic acids; iv) providing in saidreaction vial a detection reagent mixture; v) performing anamplification reaction; and vi) detecting the amplified nucleic acids,wherein steps v) and vi) are performed in said vial without removingsaid matrix from the vial.

More specifically the present invention relates to a method foranalyzing a target nucleic acid in a biological sample, wherein theamplification and detection steps are performed in sealed reaction vialswithout adding or removing any reagents or other components after theamplification step.

The present invention further relates to a method wherein said sample isapplied onto the matrix immediately prior to providing the matrix intosaid reaction vial, without drying and/or storage.

The present invention further relates to a method wherein said matrix isprovided in the reaction vial prior to applying the sample directly ontothe matrix.

The present invention further relates to a method wherein at least partof said amplification reagents and detection mixtures are provided indried form prior to the addition of the sample, and said methodincluding an optional further step of adding water prior to performingthe amplification step.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an integrated method for analyzingnucleic acids contained in a biological sample, comprising a simplifiedsample pre-treatment, amplification of the target nucleic acid to beanalyzed, and detection of said target sequence. Due to the simplifiedsample pre-treatment, the method allows automation and integration ofsample preparation, amplification and analysis of nucleic acids.

The present invention is based on the surprising finding that theamplification and detection steps may be performed on samples collectedand, optionally, stored on solid media without prior extraction, washingor elution of the nucleic acids. Only a short incubation to inactiveinhibitors and to release the nucleic acids is needed.

Inactivation of inhibitors and denaturation of disturbing proteins isachieved by use of solid matrices based on any porous or absorbingmaterial, e.g. cellulose based filter paper or wadding, cotton wad orfabric, or synthetic plastic material. Suitable matrices for use in themethod according to the present invention include absorbent materialswhich bind proteinaceous material, but do not bind nucleic acidsirreversibly, and which are impregnated with e.g., a base, an acid,reducing agents, chaotropic salts, detergents, or other agents todenature natural inhibitors within the sample. Solid matrix materialsuitable for use in the present invention may be provided as flatsheets, swabs, tablets, pellets or beads, or as a mesh or lattice.

Such solid matrix materials are readily available, and include e.g. thepaper matrix provided by Schleicher&Schuell under the trade marks 903™Specimen Collection paper or IsoCode® for collecting, storing,transporting and purifying DNA.

In a preferred embodiment of the present invention the biological sampleis provided on such an absorbent matrix. At least a portion of thematrix containing the sample is transferred to the reaction vial,together with reagents for amplification and detection. The reactionvial is then sealed and moved to a device for performing theamplification reaction. The DNA of the sample is released by a briefheat-treatment. Optionally, if the reagents are not heat stable, e.g. incase of isothermal amplification, the DNA release may be performed atambient temperature. Surprisingly, after amplification the detection maybe performed without opening the reaction vial or removing said matrix.

It has also surprisingly been found, that the sample may be applied ontothe matrix immediately prior to use. Thus, in a preferred embodiment ofthe present invention, the sample is applied onto a suitable solidmatrix, whereafter at least a portion of the matrix is transferred tothe reaction vial, without prior drying of the matrix. After addition ofnecessary reagents, the amplification and detection steps are performed.

It has further, surprisingly, been found, that the solid matrix may beprovided in the reaction vial and that the sample may be applieddirectly onto the matrix, and necessary reagents added, once the samplehas been applied. Thereafter the amplification and detection steps areperformed in the sealed vial.

The term “amplification” is meant to include any method for amplifyingnucleic acids known in the art, either thermal cycling methods such aspolymerase chain reaction (PCR; U.S. Pat. No. 4,683,202), reversetranscriptase PCR, (U.S. Pat. No. 5,310,652), and ligase chain reaction(LCR; U.S. Pat. No. 5,185,243), and any variations thereof, orisothermal methods, such as Q-Beta replicase technology (U.S. Pat. No.4,786,600), nucleic acid sequence based amplification (NASBA; U.S. Pat.No. 5,409,818), transcription mediated amplification (TMA; U.S. Pat.Nos. 5,399,491), strand displacement amplification (SDA; U.S. Pat. No.5,455,166) and multiple displacement amplification (MDA; U.S. Pat. No.6,124,120). One preferred method of amplification is asymmetric PCR,described by Innis et al., PNAS 85(24), 1988: 9436-40, which generatessingle stranded amplification products.

Other known amplification methods may also be useful in the integratedanalysis method according to the present invention. The amplificationreagents and the amplification procedure may thus vary according to themethod of choice.

The detection step is performed by any suitable detection method ofchoice. Thus the detection reagents may vary according to the method ofchoice. Suitable detection methods are based on e.g. intercalatingreagents to detect the accumulation of amplification product or toperform melting temperature analysis, or labelled primers, or specificprimer extension in which directly or indirectly labelled nucleotidesare incorporated to the amplification product, or methods in which thedirectly or indirectly labelled detection probes are degraded duringextension, or hybridization with directly or indirectly labelled probes.The detection may be based on e.g. fluorescence resonance energytransfer, fluorescence quenching or environmentally sensitive labels.Suitable detection methods may also include imaging of array of spots orbeads by confocal scanning or evanescent wave. The detection step ispreferably performed in a closed-tube format without removing the matrixor unreacted components, in order to avoid post-amplification handling.

One preferred detection method is based on hybridization using labelledprobes that recognize the amplified target nucleic acids. In such amethod, the hybridization, and thus labelling of the target nucleicacids, is achieved simply by lowering the temperature afteramplification to hybridization temperature, if necessary, whereafter theun-reacted labelled probe is quenched by a complementary probe.Detection of formed labelled hybrids may be performed on the reactionvial, without removing the matrix or other unreacted components. Thusthe reaction vials remain unopened after performing the amplificationreaction.

In a preferred embodiment of the present invention, the method isfurther simplified in a way that at least part of the reagents necessaryfor performing the amplification and detection steps, is provided in thereagent vial in dried form prior to the addition of the sample to beanalyzed. Optionally, the absorbent matrix may also be provided in thereaction vial. Such a simplified, integrated assay format minimizes themanual handling, is easily automated and decreases the risk ofcontamination, as the reaction vials may be sealed immediately afterproviding the sample and, if necessary, adding water.

A variety of samples can be analyzed using the methods of the inventionfor preparing a sample for molecular analysis. Such samples includebodily fluids, such as whole blood, saliva, sputum, urine, faecal,peritoneal and pleural fluids; lavations, such as bronchoalveolar,nasal, cervical and intestinal samples; aspiration or biopsy samples;cell cultures or microbial cultures. Specific non-limiting examplesinclude whole blood and saliva samples. The biological samples may alsobe samples taken from food or environmental samples. In addition tocomplex biological samples, the method of the present invention isequally suitable for amplifying and analysing pre-treated, purifiedsamples, for example samples where DNA and/ or RNA has already beenextracted and thereafter stored on matrix.

In a preferred embodiment, the method of the present invention iscarried out as follows. The biological sample, such as whole blood, isprovided dried on an absorbent matrix sheet or stick, or, optionally,applied to such a matrix by the user, and is allowed to dry onto thematrix. After drying, at least a portion is transferred to the reactionvial. The amplification reagents, comprising primers, nucleotides,polymerase and necessary buffers are added, as well as the detectionmixture, comprising directly or indirectly labelled nucleotides orprobes, and optionally secondary labels, or intercalators. The vial isthen sealed and moved to a thermal cycler. The DNA of the samples isreleased by a brief heat-treatment. The amplification proceeds after thepreheating. After amplification, the presence and/or composition of thenucleic acid is detected through the seal on the vial without removingthe matrix or even opening the sealed vials at any stage.

In another, highly preferred embodiment, the method of the presentinvention is carried out as a multi-vial assay. The method is performedusing a kit containing dried reagents for amplification and analysis, aswell as an absorbent matrix, either as a disk at the bottom of thevials, or as a mesh at least partly filling the vials. The sample to beanalyzed is applied, without pre-treatment, directly onto the matrix inthe reaction vial. Water is then dispensed into the vials and the vialsare sealed and moved to a thermal cycler. The DNA of the samples isreleased by a brief heat-treatment. The amplification proceeds after thepre-heating. After amplification, the presence and/or composition of thenucleic acid is then detected through the seal of the vials, withoutremoving the matrix. A suitable, readily available format for a kitaccording to the present invention is e.g. a multi-well plate.

The following examples are given to further illustrate preferredembodiments of the present invention, but are not intended to limit thescope of the invention. It will be obvious to a person skilled in theart that, as the technology advances, the inventive concept can beimplemented in various ways. The invention and its embodiments are notlimited to the examples described above but may vary within the scope ofthe claims.

EXAMPLES Example 1

Release Of Nucleic Acids From Dried Sample Disks

This example shows how the elution of dried blood and saliva disksrecommended by manufacturer can be simplified. The recommended pre-washis omitted and it is shown that the release of nucleic acids can beperformed effectively also at room temperature. The release is automatedby use of filter plates and automated filtration.

First, whole blood and saliva was collected from a volunteer. Thesamples were applied onto IsoCode® (Schleicher & Schuell) collectionpaper and allowed to dry. For analysis, eight 1.5-mm disks were punchedand the nucleic acids were eluted according to manufacturer'sinstructions, i.e. pre-wash with 500 μl of distilled water followed byincubation at +95° C. for 30 min in 135 μl of distilled water. Anothereight disks were incubated without pre-wash at +95° C. for 30 min. Afterincubation, the supernatant was analysed. The automated release wasdemonstrated by punching a 1.5-mm disk together with 40 μl of distilledwater into a vial in a 96-well filtration plate, and the samples wereincubated at room temperature for 30 min. After incubation, the filterplate was aligned with reaction plate and the supernatant wastransferred into the reaction plate by pressure from top of the filterplate.

The closed-tube PCR amplification and detection reaction was performedin a total volume of 50 μl and the final concentration in the reactionmixture was following: 1× DyNAzyme™ buffer (Finnzymes), 0.2 mM dNTP's,2.5 mM MgCl2, 0.1 μM CFTR exon 10 forward primer (5′ AAG CAC AGT GGA AGAATT TC 3′), 0.1 μM CFTR exon 10 reverse primer (5′ CTC TTC TAG TTG GCATGC T 3′), 0.02 U/μl DyNAzyme™ enzyme (Finnzymes), 20 nM analytespecific oligonucleotide probe (5′ TAA AGA AAA TAT CAT CTT TGG TGT TTCCTA TAA 3′) labelled at its 5′ end with a stable fluorescent W14054 Tbchelate (Wallac) and 0.4 μM quencher probe complement to the Tb probe(5′ ATG ATA TTT TCT TTA 3′) labelled at its 3′ with B HQ1 (Bio-searchTechnologies), 10 μl of supernatant from the 95° C. incubation, and as acontrol 0.4 ng/μl genomic DNA sample purified from a volunteer's wholeblood. The supernatant from the automated release was analysed by adding10 μl of the reaction mixture to the final concentration describedabove.

The PCR program was as follows: Pre-heating +95° C. 1 min; 35 cycles offollowing +95° C. 30 s, ramping from +95° C. to +84° C. 0.2° C. s,ramping from +84° C. to +56° C. 2.5° C./s, +56° C. 1 min, +66° C. 1 minand final extension 7 min. After amplification, the reaction wasincubated at +30° C. for 5 min, whereafter the time-resolvedfluorescence was measured with Victor2 1420 Multilabel Counter (Wallac).

The results of this experiment are shown in FIG. 1 by the average andstandard deviation of time-resolved Tb fluorescence of each reaction induplicates. Negative and positive PCR controls are drawn by bars A andB, respectively. When the simplified release (D and E) is compared toelution by manufacturer's recommendation (C), it is clearly seen thatthe analysis can be performed with blood (black bars) and saliva (greybars) disks without pre-wash both at room temperature (D) and at +95° C.(E). The result from the supernatants is also comparable to theestimated amount of genomic DNA per blood disk.

Example 2

DNA Analysis From Whole Blood And Saliva

This example shows how the pre-treatment of blood and saliva samples canbe simplified to enable simple automation. The sample is collected ontoprotein denaturing matrix, and the release of nucleic acids from thematrix, amplification and detection are all performed in a singlereaction vial. After release, the amplification and detection reagentsare added and the reaction vial is sealed, and after amplification andshort analytical incubation, the fluorescence is measured withoutopening the reaction vial, i.e., without removal of the matrix.

First, whole blood and saliva was collected from two volunteers. Thesamples were applied onto IsoCode® (Schleicher & Schuell) collectionpaper and allowed to dry. For analysis, a 1.2-mm blood disk or two1.2-mm saliva disks were punched and the nucleic acids were released atroom temperature for 30 min in 40 μl of distilled water. The release wasperformed in PCR vials and as a reference in separate micro tubes. Afterrelease, the PCR/detection reaction mixture was added into the PCR vialcomprising the disk. As reference, the 40-μl supernatant from theseparate tubes was analysed.

The closed-tube PCR amplification and detection reaction was performedin a total volume of 50 μl. A 10-μl portion of following PCR/detectionreaction mixture was added into the PCR: 5× DyNAzyme™ buffer(Finnzymes), 1 mM dNTP's, 12.5 mM MgCl2, 1 μM CFTR exon 10 forwardprimer (5′ AAG CAC AGT GGA AGA ATT TC 3′), 0.25 μM CFTR exon 10 reverseprimer (5′ CTC TTC TAG TTG GCA TGC T 3′), 0.1 U/μl DyNAzyme™ enzyme(Finnzymes), 83 nM analyte specific oligonucleotide probe (5′ ACC AAAGATGAT ATT TAA A 3′) labelled at its 5′ end with a stable fluorescentW8184 Eu chelate (Wallac) and 166 nM quencher probe complement to the Euprobe (5′ TCA TTG GTG TTT 3′) labelled at its 3′ with BHQ1 (BiosearchTechnologies). As a control, 20 ng/reaction of genomic DNA samplepurified from a volunteer's whole blood was analysed.

The PCR program was as follows: Pre-heating 95° C. 1 min; 35 cycles offollowing +95° C. 30 s, +56° C. 1 min, +66° C. 1 min, final extension 7min and final denaturation 8 min. After amplification, the reaction wasincubated at +40° C. for 20 m in and at +22° C. for 15 min, whereafterthe time-resolved fluorescence was measured with Victor2 1420 MultilabelCounter (Wallac) directly from the unopened vial.

The results of this experiment are shown in FIG. 2 by the average andstandard deviation of time-resolved Eu fluorescence of each reaction intriplicates. Negative and positive PCR controls are drawn by bars A andB, respectively. C-D represent results from blood and E-F from salivasamples obtained from 2 volunteers. When the integrated release in thereaction vial without removal of matrix at any point (hatched bars) iscompared to release in a separate vial (grey bars), it is clearly seenthat the whole analysis can be performed with blood disk and salivadisks without removal of disk. The result shows that the release fromthe matrix can be performed at room temperature in the reaction vial andafter amplification the detection can be performed without removal ofmatrix enabling simple automation of whole analysis.

Example 3

Integrated DNA Analysis From Whole Blood And Saliva

This example shows how the release of nucleic acids from matrix can befurther simplified to enable simple automation. The sample is collectedonto protein denaturing matrix, and the release of nucleic acids,amplification and detection are all performed in a single reaction vialeven in the presence of amplification and detection reagents. The matrixcomprising the sample is added together with the amplification/detectionreagents into a reaction vial, the vial is sealed, and afteramplification and short analytical incubation, the fluorescence ismeasured without opening the reaction vial, i.e. without removal of thematrix.

First, whole blood and saliva was collected from two volunteers. Thesamples were applied onto IsoCode® (Schleicher & Schuell) collectionpaper and allowed to dry.

For analysis, a 1.2-mm blood disk or two 1.2-mm saliva disks werepunched into PCR vials. The closed-tube PCR amplification and detectionreaction was performed in a total volume of 50 μl in followingPCR/detection reaction mixture: 1× HotStarTaq® buffer (Qiagen), 0.2 mMdNTP's, 2.5 mM MgCl2, 0.05% BSA, 0.2 μM CFTR exon 10 forward primer (5′AAG CAC AGT GGA AGA ATT TC 3′), 0.05 μM CFTR exon 10 reverse primer (5′CTC TTC TAG TTG GCA TGC T 3′), 0.02 U/μl DyNAzyme™ enzyme (Finnzymes),17 nM analyte specific oligonucleotide probe (5′ ACC AAA GAT GAT ATT TAAA 3′) labelled at its 5′ end with a stable fluorescent W8184 Eu chelate(Wallac) and 33 nM quencher probe complement to the Eu probe (5′ TCA TTGGTG TTT 3′) labelled at its 3′ with BHQ1 (Biosearch Technologies). As acontrol, 20 ng/reaction of a volunteer's genomic DNA sample on disk wasanalysed. As a reference, the release of nucleic acids was performed atroom temperature for 30 min in 40 μl of distilled water, and analysed inthe closed-tube PCR/detection after addition of a 10-μl portion ofreaction mixture as 5× concentrate.

The PCR program was as follows: Pre-heating +95° C. 15 min; 35 cycles offollowing +95° C. 30 s, +56° C. 1 min, +66° C. 1 min, final extension 7min and final denaturation 8 min. After amplification, the reaction wasincubated at +40° C. for 20 min and at +22° C. for 15 min, whereafterthe time-resolved fluorescence was measured with Victor2 1420 MultilabelCounter (Wallac).

The results of this experiment are shown in FIG. 3 by the average andstandard deviation of time-resolved Eu fluorescence of each reaction intriplicates. Negative and positive PCR controls are drawn by bars A andB, respectively. C-D represent results from blood and E-F from salivasamples obtained from 2 volunteers. When the release inamplification/detection reagents (grey bars) is compared to release indistilled water (white bars), the result is nearly equal with bothmethods. The result shows that release from the matrix can be performedeven in the amplification/detection mixture, and the whole process canbe performed in a single vial in a closed-tube format enabling simpleautomation of whole analysis.

Example 4

Integrated Sample Pre-Treatment And DNA Analysis From Whole Blood

This example shows how the matrix comprising the sample can be used inanalysis right after the application of the sample enabling developmentof amplification/detection devices comprising the matrix in the reactionvial. The sample is applied onto the protein denaturing absorbent matrixin the reaction vial, the amplification/detection reagents are added,the vial is sealed, and after amplification and short analyticalincubation, the fluorescence is measured without opening the reactionvial.

The closed-tube PCR amplification and detection reaction was performedin a total volume of 50 μ. First, a 1.2-mm disk from IsoCode®(Schleicher & Schuell) collection paper was punched into PCR vials.Then, a 0.35-μl sample from a volunteer's whole blood was applied intothe vial. After various time points, a 50-μl portion of PCR/detectionreaction mixture (as follows) was added: 1× Phusion™ HF buffer(Finnzymes), 0.2 mM dNTP's, 2.5 mM MgCl2, 0.02% BSA, 0.2 μM CFTR exon 10forward primer (5′ AAG CAC AGT GGA AGA ATT TC 3′), 0.05 μM CFTR exon 10reverse primer (5′ CTC TTC TAG TTG GCA TGC T 3′), 0.02 U/μl DyNAzyme™enzyme (Finnzymes), 17 nM analyte specific oligonucleotide probe (5′ ACCAAA GAT GAT ATT TAA A 3′) labelled at its 5′ end with a stablefluorescent W8184 Eu chelate (Wallac) and 33 nM quencher probecomplement to the Eu probe (5′ TCA TTG GTG TTT 3′) labelled at its 3′with BHQ1 (Biosearch Technologies). As a control, 20 ng/reaction of avolunteer's genomic DNA sample on disk was analysed.

The PCR program was as follows: Pre-heating +95 C. 15 min; 35 cycles offollowing +95° C. 30 s, +56° C. 1 min, +66° C. 1 min, final extension 7min and final denaturation 8 min. After amplification, the reaction wasincubated at +40° C. for 20 min and at +22° C. for 15 min, whereafterthe time-resolved fluorescence was measured with Victor2 1420 MultilabelCounter (Wallac).

The results of this experiment are shown in FIG. 4 by the average andstandard deviation of time-resolved Eu fluorescence of each reaction in4 replicates. Negative and positive PCR controls are drawn by bars A andB, respectively. C-F represent results from blood samples analysed. Thesignal from the analysis which has been performed right after applyingthe sample into the amplification/detection vial comprising the matrix(F) is equal to those which have been let dry 1, 2 and 16 h (C-E,respectively). The result shows that the whole process can be performedin a single vial avoiding multiple steps from sample pre-treatment todetection allowing very simple automation.

Example 5

Integrated DNA Analysis From Whole Blood Using Dried Reagents

This example shows the extreme simplification of the nucleic acidanalysis process from sample pre-treatment to detection. The reagentsare dried into the reaction vial, and the sample is either applied ontothe protein denaturing absorbent matrix in the vial, or the matrixcontaining sample is punched into the vial together with water. Then thevial is sealed, and after amplification and short analytical incubation,the fluorescence is measured without opening the vial.

First, a 12.5-μl portion of following amplification/detection reactionmixture was allowed to dry in the reaction vial at room temperature for2 h: 4× Phusion™ HF buffer (Finnzymes), 0.8 mM dNTP's, 10 mM MgCl2,0.08% BSA, 0.8 μM CFTR exon 10 forward primer (5′ AAG CAC AGT GGA AGAATT TC 3′),0.2 μM CFTR exon 10 reverse primer (5′ CTC TTC TAG TTG GCATGC T 3′), 0.08 U/μl DyNAzyme™ enzyme (Finnzymes), 66 nM analytespecific oligonucleotide probe (5′ ACC AAA GAT GAT ATT TAA A 3′)labelled at its 5′ end with a stable fluorescent W8184 Eu chelate(Wallac) and 133 nM quencher probe complement to the Eu probe (5′ TCATTG GTG TTT 3′) labelled at its 3′ with BHQ1 (Biosearch Technologies).

Then, the reaction vial comprising the dried reagents was used in theclosed-tube PCR amplification and detection reaction in a total volumeof 50 μl. A 1.2-mm blood disk of previously collected volunteer's sampleon IsoCode® (Schleicher & Schuell) was punched into the reaction vialwith simultaneous dispensing of 50 μl of distilled water. Or, a 1.2-mmdisk from IsoCode® (Schleicher & Schuell) collection paper was punchedinto the vial, and then, a 0.35-μl sample from a volunteer's whole bloodwas applied into the vial, and 50 μl of distilled water was added. Also,supernatant after release of nucleic acids in 50 μl of distilled waterin a separate vial at room temperature for 30 min was used as sample inanalysis. As a positive control, 20 ng/reaction of a volunteer's genomicDNA sample on disk was analysed.

The PCR program was as follows: Pre-heating +95° C. 15 min; 35 cycles offollowing +95° C. 30 s, +56° C. 1 min, +66° C. 1 min, final extension 7min and final denaturation 8 min. After amplification, the reaction wasincubated at +40° C. for 20 min and at +22° C. for 15 min, whereafterthe time-resolved fluorescence was measured with Victor2 1420 MultilabelCounter (Wallac).

The results of this experiment are shown in FIG. 5 by the average andstandard deviation of time-resolved Eu fluorescence of each reaction in4 replicates. Negative and positive PCR controls are drawn by bars A andB, respectively. C-E represent the results from blood samples obtainedfrom 3 volunteers, respectively. When the integrated release in thereaction vial (hatched bars) is compared to the release in a separatevial (grey bars), it is clearly seen that the whole analysis can beperformed using dried reagents even without removal of disk. Also, Eshows that the matrix can be integrated into the reaction vialcomprising the dried reagents, and the sample can be applied rightbefore the analysis. The result shows that the whole process from samplepre-treatment to detection can be performed in a single reaction vialcomprising all reagents dried enabling extremely simple automation.

1. A method for analyzing a target nucleic acid in a biological sample,comprising the following steps i) providing an absorbent matrix in areaction vial; ii) providing a biological sample containing said targetnucleic acids on, or applying it onto, said absorbent matrix; iii)providing in said reaction vial a reagent mixture for amplifying saidnucleic acids; iv) providing in said reaction vial a detection reagentmixture; v) sealing said reaction vial; vi) performing an amplificationreaction; and vii) detecting the amplified nucleic acids, wherein stepsvi) and vii) are performed in said vial without removing said matrixfrom the vial, and wherein the detection step is performed inclosed-tubes, without opening the seals.
 2. The method according toclaim 1, wherein the sample is provided on the absorbent matrix, and atleast a portion of said matrix is provided into the vial together withsaid sample.
 3. The method according to claim 2, wherein the sample isapplied onto the matrix immediately prior to providing said matrix intothe reaction vial, without drying and/or storage.
 4. The method ofclaims 1, wherein the matrix is provided in the reaction vial prior tostep ii) and the sample is applied onto said matrix contained in saidvial.
 5. The method according to claims 1, wherein the matrix is anabsorbent material that does not irreversibly bind nucleic acids.
 6. Themethod according to claim 5, wherein the matrix comprises a proteinbinding and/or a protein denaturing agent.
 7. The method according toclaim 5, wherein the matrix comprises a chaotropic agent.
 8. The methodaccording to claims 1, wherein at least part of the amplification anddetection reagent mixtures are contained in the reaction vial in driedform, said method including an optional further step of adding waterprior to step v).
 9. The method according to claims 1, wherein theamplification is performed by PCR.
 10. The method according to claims 1,wherein the detection step is performed by hybridization of theamplified sample to a fluorescently labelled probe.
 11. A kit for use ina method according to claims 1, comprising at least one reaction vialcomprising an absorbent matrix; a reagent mixture for amplifying saidnucleic acids; and a detection reagent mixture for detecting saidamplified nucleic acids; wherein at least part of said reagent anddetection mixtures are provided in dried form.
 12. A kit according toclaim 11, wherein all reagents are provided in dried form in saidreaction vial.